Project 2 - Abstract Modification of autologous T-cells with chimeric antigen receptor (CAR) molecules has revolutionized the treatment of many leukemias, and is designed to enable ?plug and play? targeting of any surface-expressed marker of human disease. We are interested in optimizing CAR-T therapies for persistent HIV-1 infection. Importantly, although the list of hematological malignancies to which CAR-T can be applied is rapidly expanding, several barriers have prevented application to HIV+ individuals. First and foremost, CAR-T function is frequently downregulated or lost upon migration to tissues, for example limiting targeting of solid tumors. This also represents a key limitation for targeting of latently HIV-1 infected cells that reside at sites including lymph nodes, gut, and the brain. Furthermore, increasing evidence suggests that in order for CAR-T to recognize their cognate targets, a threshold level of antigen expression may be required at the target cell surface. The central goal of our U19 consortium is to understand the fate of a CAR T-cell in vivo. In our project, we will compare virus-specific CARs to CARs directed against CD20+ leukemias, mechanisms of action in antigen-rich vs. antigen-sparse environments, and the ability of CAR-T to maintain potent, target-specific function after migrating to secondary tissue sites known to harbor latent virus. We will address these questions in our well-established nonhuman primate (NHP) model of suppressed HIV-1 infection, focusing on the optimized CD4CAR molecule developed by Dr. Riley in Project 3. We will first compare CD4CAR-T to a previously-characterized NHP version of the successful, leukemia-targeting CD20 CAR molecule (?NHP CD20 CAR-T?), in 6 uninfected animals. Next, we will focus on CD4CAR-T in 12 animals that have been previously infected with simian/human immunodeficiency virus (SHIV) and suppressed by antiretroviral therapy (ART). Our studies in a total of 18 uninfected or infected, suppressed animals will provide unprecedented insights into the mechanisms that promote engraftment, persistence, and function in vivo, and/or lead to silencing or inhibition of antigen-dependent expansion. We have chosen the NHP model for our studies, as a key aspect of our approach is to better understand CAR-T trafficking and function in tissues. Using well-established assays to measure tissue resident memory T-cells (TRM), along with immunohistochemistry and transcriptional profiling approaches, we will characterize CD4CAR-T function in low and high antigen environments (i.e. before and after release of ART), and ii) benchmark these activities against our positive control, NHP CD20 CAR-T. Our NHP research aims will apply the gold standard for in vivo modeling of suppressed HIV infection, and are highly complementary to experiments proposed in Project 1/Wherry, Project 3/Riley, and Project 4/Coughlin and Tebas.